KR102013731B1 - Method for stabilising suspensions of red blood cells encapsulating an active ingredient, the suspensions obtained - Google Patents

Abstract

A method of obtaining a stabilized suspension of RBC encapsulating the active ingredient from a sewn red blood cell (RBC) comprising the active ingredient, the method comprising the re-incubation in an incubation solution for at least 30 minutes at an osmotic pressure of at least 280 mOsmol / kg. Incubated the sealed RBCs, the incubation solution being a solution that does not contain an agent that denatures the RBC membrane, thereafter removing the liquid medium from the incubated suspension, and obtaining the RBCs from the suspension in a patient. Suspending in a solution to enable injection. The suspensions obtained are, in particular, extracellular hemoglobin levels and / or 2% or less, maintained at or below 0.5 g / dl, in particular at most 0.2 g / dl, at a temperature of 2 to 8 ° C. at 72 hours after suspension in a preservation solution, In particular, hemolysis rate is maintained at 1% or less.

Description

Method for stabilising suspensions of red blood cells encapsulating an active ingredient, the suspensions obtained

The present invention relates to a method of stabilizing a suspension of red blood cells that encapsulates an active ingredient. The present invention also relates to a process for preparing such suspensions, to treatment methods using the suspensions and to novel stable suspensions of red blood cells encapsulating the active ingredient.

Several methods have been described that allow the incorporation of the active ingredient into red blood cells. Of these methods, the so-called lysis-resealing technique is most widely used. The technique involves three variants, hypotonic dialysis, hypopotent pre-swelling and hypotonic dilution, all based on the difference in the osmotic pressure between the internal and external erythrocytes. These modifications commonly have the following five steps: washing and centrifuging blood cell residues one or more times in physiological buffers, leaving red blood cells in contact with the storage liquid medium, causing the opening of pores in the red blood cell membrane, The active ingredient enters the red blood cells and the active ingredient is enclosed inside the red blood cells by suspending (resealing) the pores with hypertonic buffer and then suspended in a preservation solution. The hypotonic dialysis method is the most advantageous and subject to industrial development. The method described in EP 1 773 452 is presently the best performance method and has the advantage of being renewable and improving the encapsulation yield of the active ingredient.

Thus, the stability of the product obtained is a key factor in its use in the treatment of humans. In particular, the amount of extracellular hemoglobin contained in the product at the time of injection into the patient should be below a predetermined threshold. For example, the limit required by the US Food and Drug Administration for extracellular hemoglobin is 0.2 g / dL or less in the final product used for human injection.

The product obtained by the methods of the prior art undergoes hemolysis during its storage period and transportation before injection. This hemolysis, caused by the most fragile erythrocyte rupture, releases hemoglobin into the extracellular medium, which results in no longer meeting FDA requirements at the time the product is injected.

Therefore, it is an object of the present invention to propose a method which makes it possible to improve the stability of erythrocyte suspensions that encapsulate the active ingredient.

One specific object of the present invention is a method for making a suspension of erythrocytes comprising an active ingredient and having stable extracellular hemoglobin levels after storage, or maintained according to recommendations provided by the FDA or other health authorities. Is to suggest.

It is yet another object of the present invention to propose such a method, which is applicable to suspensions of red blood cells encapsulating the active ingredient, in particular irrelevant to the method used for its preparation, using the dissolution-sealing method.

It is a further object of the present invention to propose such a method which enables the production of red blood cell suspensions which encapsulate the active ingredient with a stable and high cell yield.

In order to obtain a suspension comprising the largest possible proportion of hemolytically resistant red blood cells, these objectives are achieved by removing the most fragile red blood cells from the encapsulation method, ie the suspension resulting from post-resealing. , And other purposes can be reached. Therefore, the suspension of erythrocytes can be preserved without undergoing significant hemolysis until injected into the patient, making the suspension with low levels of extracellular hemoglobin available at the time of injection. Therefore, the method of the present invention enables the removal of the most fragile red blood cells among the extracellular hemoglobin and the extracellular active ingredient. Applicants succeeded in achieving this while finding a good compromise between maintaining good cell yield, removing the most fragile red blood cells and maintaining the maximum number of red blood cells. Applicants have also been able to surprisingly determine the conditions that improve cell yield while allowing the stabilization of suspensions of red blood cells encapsulating the active ingredient.

By << encapsulating >> it is meant that the active ingredient is contained essentially or completely inside. << essentially >> means that a small proportion of the active ingredients can nevertheless be trapped in the membrane.

By << encapsulating an active ingredient >> the erythrocyte comprises a molecule having the active ingredient function, or an assembly formed by the erythrocyte and the molecule it contains is responsible for the active ingredient function. It means to have.

By << incubation solution >> is meant a solution containing red blood cells that encapsulates the active ingredient during the incubation step. Incubation can be carried out over a wide range of hematocrit, especially in the range from 10 to 85% hematocrit.

By << preservation solution >> is meant a solution placed in suspension in a form suitable for storage until stabilized red blood cells which encapsulate the active ingredient are used. The preservation solution preferably comprises one or more agents which promote the preservation of red blood cells, in particular selected from glucose, dextrose, adenine and mannitol.

When the suspension is stored at 2-8 ° C. by <fragile red blood cells >>, it is easy to dissolve once in suspension in a preservative solution, especially after 1 to 72 hours. Refers to red blood cells derived from the process.

By << initial haematocrit >> means hematocrit before cell loss due to lysis of erythrocytes that are susceptible to breakage during incubation.

With the stability over time of the red blood cells comprising the active ingredient, the concept of <stabilisation> is essential, in particular in view of the loss of intracellular hemoglobin or extracellular hemoglobin levels.

With <stabilised suspension of red blood cells >>, as a use in humans, such use may be from 1 to 72 after the production of a batch of red blood cells, possibly including the active ingredient. Particularly meant suspensions with extracellular hemoglobin levels maintained below 0.5 g / dL, in particular below 0.3 g / dL, preferably below 0,2 g / dL, until use which occurs over time. It may also be characterized by a hemolysis rate that is maintained at 2% or less, in particular 1.5% or less, preferably 1% or less, at a temperature of 2 to 8 ° C. in a 72 hour time difference.

By << ready-to-use stabilized suspension of red-blood cell >> is meant a stabilized suspension in a solution, in particular a preservative solution, which is acceptable for injection by the patient. Its hematocrit is typically at least 40%.

By << residue of red blood cells >> or packed red blood cells, it refers to a concentrate of red blood cells collected after separation of red blood cells from a liquid medium that was previously suspended therein. Separation may be performed by filtration or centrifugation. Centrifugation is a commonly used means for such separation. The residue comprises a certain proportion of the liquid medium. Typically, the hematocrit of the residue is 70-85%.

This method can be applied regardless of the technique used to contain or encapsulate the active ingredient. It is most particularly applicable to the methods described in EP 1 773 452, which can be referred to by those skilled in the art, using leading dissolution-sewing techniques, in particular hypotonic dialysis. The content of EP 1 773 452 is hereby incorporated by reference. As will be appreciated by reading the following description, the stabilization method of the present invention may be applied to a suspension or residue of sewn red blood cells encapsulating the active ingredient, or the suspension or residue of sewn red blood cells is Dissolution and sewing steps may be achieved until obtained.

Accordingly, the present invention aims at a method for obtaining a stabilized suspension of red blood cells (RBCs) that encapsulates an active ingredient from sewn red blood cells comprising the active ingredient. The method comprises incubating the sewn red blood cells in an osmotic incubation solution of at least 280 mOsmol / kg, in particular from about 280 to about 380 mOsmol / kg, preferably from about 290 to about 330 mOsmol / kg. Incubation is particularly carried out for a time of at least 30 minutes, in particular for a time of at least 1 hour. Incubation is named post-sewing, ie it is performed on the stitched RBCs. The liquid medium is then removed from the incubated suspension and the resulting RBCs are suspended in a solution that allows injection of the suspension in the patient, preferably in a preservation solution that allows injection of the suspension in the patient.

Incubation solutions are generally salt solutions (eg, NaCl, KCl and / or phosphate based solutions) that contain ions that allow at least osmotic pressure to be controlled. It may comprise additional components, in particular carbohydrates, in particular sugars, and / or acidic and / or basic adducts which allow for the adjustment of the pH (specifically about 6 to about 8.5, preferably about 7 to about 7.5). . Incubation solutions are agents which denature the RBC membrane, for example bridging or cross-linking chemical agents, for example bis (sulfosuccinimidyl) suverate (BS3), glutaraldehyde And neuroamidase. Thus, it is an inert incubation solution or a solution that does not fragilise the membranes of sewing RBCs.

In one embodiment, the method comprises, prior to incubation, encapsulating the active ingredient with RBC to encapsulate and obtaining a stitched RBC comprising the active ingredient.

Preferably, washing of the stitched RBCs (at least one wash cycle) is performed before incubation.

The stitched RBCs applied to this method may be a suspension of RBCs in a preservation solution. It is then possible to dilute the stock solution by incubation solution to obtain an osmotic pressure of the value according to the invention. Alternatively, the RBCs can be separated from the stock solution, for example by centrifugation or filtration, after which the incubation solution is added. It is also possible to wash the stitched RBCs (as described below, one or more cycles); The wash cycle preferably includes dilution of the suspension prior to suspension in the incubating solution, followed by separation. In addition, the stitched RBCs to which this method is applied may be a suspension of the stitched RBCs still present in the stitching solution. Thereafter, it is possible to separate the RBCs from the sewing solution, for example by centrifugation or filtration, and / or to wash the sewing RBCs (one or more cycles, as described later); The wash cycle preferably includes dilution of the suspension prior to suspension in the incubating solution, followed by separation.

Thus, the method comprises placing and incubating the sewn red blood cells in an osmotic incubation solution of at least 280 mOsmol / kg, in particular about 280 to about 380 mOsmol / kg, preferably about 290 to about 330 mOsmol / kg. Include.

The subject of the invention is in particular the encapsulation of the active ingredient via dissolution-sewing inside the RBC, to obtain a suspension or residue containing the stitched RBC comprising the active ingredient, washing of the stitched RBC (Washing Cycle 1 At least 280 mOsmol / kg, and then placing it in an osmotic incubation solution of at least 280 mOsmol / kg, in particular from about 280 to about 380 mOsmol / kg, preferably from about 290 to about 330 mOsmol / kg, and incubation Is a method for obtaining a stabilized suspension of red blood cells (RBC) that encapsulates. Incubation is particularly carried out for a time of at least 30 minutes, in particular for a time of at least 1 hour. The liquid medium is then removed from the incubated suspension and the resulting RBCs are suspended in a solution that allows injection of the suspension in the patient, preferably in a preservation solution that allows injection of the suspension in the patient. The osmotic pressure indicated is the osmotic pressure of the solution in which the RBC is present in suspension or in the residue at the point under consideration.

In particular the method according to the invention comprises the following steps:

(a) contact with a storage medium (which enables the opening of the pores in the membrane of the RBC), contact with the active ingredient (which allows access to the RBC), in particular isotonic or hypertonic medium, advantageously high Encapsulating the active ingredient inside the RBC, including sewing the RBC with the medium;

(b) a suspension or residue comprising an RBC comprising the active ingredient and an osmotic solution of at least 280 mOsmol / kg, in particular from about 280 to about 380 mOsmol / kg, preferably from about 290 to about 330 mOsmol / kg Obtaining or preparing,

(c) for a time of at least 30 minutes, in particular at least 1 hour, the residue or suspension of step (b) as it is or at least 280 mOsmol / kg, in particular from about 280 to about 380 mOsmol / kg, preferably from about 290 to Incubating after addition of the incubation solution at an osmotic pressure of about 330 mOsmol / kg;

(d) removing the liquid medium from the suspension incubated in step (c),

(e) suspending the RBC obtained in (d) in a solution that allows injection of the suspension in the patient, preferably in a preservation solution that enables injection of the suspension in the patient.

According to a first aspect, the subsequent step after encapsulation by dissolution-sealing, in particular step (b), is for example at least 280 mOsmol / kg, in particular from about 280 to after the dissolution-sealing step or step (a) One or more wash cycles, preferably two or more, by dilution of a suspension of the residue comprising the sewn RBC obtained in an osmotic solution of about 380 mOsmol / kg, preferably about 290 to about 330 mOsmol / kg Three wash cycles, followed by obtaining a residue or suspension of the RBC. This residue or this suspension comprises an RBC comprising the active ingredient and an osmotic pressure of at least 280 mOsmol / kg, in particular from about 280 to about 380 mOsmol / kg, preferably from about 290 to about 330 mOsmol / kg. Then, the following steps are applied, for example, (c), (d) and (e).

Subsequent steps after dissolution-sewing, such as (b) to (e), result in dissolution of red blood cells or the majority thereof, particularly 50, 60, 70, 80 or 90%, or more, that are prone to breakage Under conditions of For this purpose it can be carried out at incubation time, incubation temperature and osmotic pressure of the solution in which the RBC is in suspension. The higher the osmotic pressure, the longer the incubation time. The lower the osmotic pressure, the shorter the incubation time to achieve the same effect. Similarly, the higher the temperature, the shorter the incubation time and vice versa. One or more wash cycles will then allow for the removal of cell debris and extracellular hemoglobin, and removal of extracellular active ingredients.

According to the invention, the wash cycle comprises dilution of a suspension or residue of the RBC, followed by separation of the RBC and the wash solution. Preferably, the washing step preferably comprises two or three dilution-separation cycles. Separation may be performed using suitable means such as filtration and centrifugation. Centrifugation is preferred. Washing with an incubation solution is preferred. Incubation is not limited by hematocrit in suspension. Therefore, it is usually possible to incubate suspensions with an initial hematocrit of 10 to 85%, in particular 40 to 80%. The term residue is used further at and after 70% and the suspension is used further below this value.

The removal step or step (d) is especially intended to << remove >> the liquid portion from the incubated suspension or residue to remove cellular debris and extracellular hemoglobin, and consequently the extracellular active ingredient.

According to the first aspect of the removal step or step (d), separation, in particular centrifugation, is carried out, which is particularly applicable to suspensions. This separation may be followed by one or more, for example two or three wash cycles by dilution in isotonic solution, and in particular subsequent separation by centrifugation.

According to a second aspect of the removal step or step (d), dilution is carried out, in particular before separation by centrifugation, which is applicable to the suspension or residue. Dilution can be carried out in particular by isotonic washing solutions or stock solutions.

In the final step or step (e), the final suspension can be prepared and administered to the patient without any other treatment.

According to a first aspect of this step, the residue of RBC derived from the removal step or step (d) is diluted by injection solution, in particular preservation solution.

According to a second aspect of this step, one or more wash cycles are carried out by dilution after dilution, using injection solutions, in particular preservation solutions, for the residue of RBC derived from the removal step or step (d). After washing, the RBCs are re-suspended in injection solutions, in particular preservation solutions.

The method of the present invention may further comprise one, several or all of the following features:

The incubation step or step (c) is carried out at a temperature of about 2 to about 39 ° C. for a sufficient time to ensure dissolution of the fragile RBCs;

The incubation step or step (c) is carried out at low temperature, in particular from about 2 to about 10 ° C., in particular from about 2 to about 8 ° C., from about 1 h to about 72 h, in particular from 6 h to about 48 h, preferably from about 19 h and about 30 h.

The incubation step or step (c) is carried out at a higher temperature of about 20 to about 39 ° C., in particular at ambient temperature (25 ° C. ± 5 ° C.) and from about 30 minutes to about 10 h, in particular from about 1 h to about 6 h Preferably, from about 2 h to about 4 h; It is possible to work at temperatures much higher than the ambient temperature, but this can have a negative effect on the yield, P50 and / or 2,3-DPG content of the cells;

In the incubation step or step (c), the suspension has an initial hematocrit of 10 to 85%, in particular 40 to 80%; It is possible to incubate, for example, residues derived from a separation with 70 to about 85% hematocrit, or diluted residues with about 40 to 70% hematocrit;

The incubation step comprises stirring the suspension;

The incubation step does not comprise stirring;

As a solution for washing and / or incubation, an aqueous NaCl solution is used at a concentration to achieve the desired osmotic pressure; For example, the solution may comprise 0.9% NaCl; In particular in addition to NaCl or another salt source (eg KCl, phosphate), this solution may also comprise glucose, in particular glucose monohydrate, monosodium phosphate dihydrate, disodium phosphate dodecahydrate; For example, the composition comprises 0.9% NaCl, 0.2% glucose monohydrate, 0.034% monosodium phosphate dihydrate, 0.2% disodium phosphate dodecahydrate;

The washing of the last step or step (e) is carried out with a preservation solution;

The osmotic pressure of the ready-to-use suspension or solution (liquid portion) in the suspension injected into the patient is about 280 to about 380 mOsmol / kg, preferably about 290 to about 330 mOsmol / kg;

At least 40% of the hematocrit of the ready-to-use suspension or suspension that can be injected in the patient;

All washing, incubation steps are carried out with a preservation solution;

The wash solution of step (b) and / or the wash solution and preservation solution of step (e) comprise one or more compounds having the same composition and promoting the preservation of red blood cells;

The preservation solution (and in the case of an applicable washing or incubation solution) is an aqueous solution containing NaCl, adenine and at least one compound from among glucose, dextrose and mannitol;

The preservation solution (and in the case of an applicable washing or incubation solution) contains NaCl, adenine and dextrose, preferably an AS3 medium;

The preservation solution (and in the case of an applicable washing or incubation solution) contains NaCl, adenine, glucose and mannitol, preferably SAG-mannitol or ADsol medium.

The invention may also be defined by a method for obtaining a stabilized suspension of RBCs comprising the active ingredient, in particular comprising the following steps:

(a) contacting the storage medium (which enables the opening of the pores in the membrane of the RBC), contacting the active ingredient (which allows its entry into the RBC), and sewing the RBC by isotonic or hypertonic medium And a so-called fragile RBC, ie a suspension of RBCs containing a group of RBCs, which are susceptible to dissolution after 1 to 72 h, especially if the suspension is stored at 2 to 8 ° C. once suspended in a preservation solution, or Encapsulating the active ingredient inside the RBC, comprising harvesting the residue,

(bc) washing and incubating the RBC obtained in (a) in solution and under fragile RBCs, or a majority thereof, especially under conditions that result in dissolution of RBC in excess of 50, 60, 70, 80 or 90%. ,

(d) removing the liquid medium from the incubated suspension in the preceding step,

(e) suspending the RBC obtained in (d) in a solution that allows injection of the suspension in the patient, preferably in a preservation solution that allows injection of the suspension in the patient.

According to a characteristic, step (b) comprises an RBC comprising the active ingredient and an osmotic pressure of at least 280 mOsmol / kg, in particular from about 280 to about 380 mOsmol / kg, preferably from about 290 to about 330 mOsmol / kg Obtaining or preparing suspensions or residues.

According to the properties, during step (c) for at least 30 minutes, in particular for at least 1 h, the residue or suspension of step (b) is left as is or at least 280 mOsmol / kg, in particular from about 280 to about 380 mOsmol / kg, preferably Comprises incubation after addition of an osmotic incubation solution of about 290 to about 330 mOsmol / kg.

According to the property, step (d) comprises washing the RBC obtained in (c), in particular two or three wash cycles, in order to remove cell debris and extracellular hemoglobin.

This method can reproduce the two embodiments described herein and various aspects and characteristics thereof.

The process of the invention in particular comprises the following steps:

(a) contacting the storage medium to open the pores in the membrane of the RBC, contacting the active ingredient to enable its entry into the RBC, and sewing the RBC with isotonic or hypertonic medium; Encapsulating the active ingredient inside the RBC. It should be noted that the active ingredient may be present in the suspension of RBC prior to its dissolution, or that the active ingredient may be added during or after dissolution but always before sewing.

In one embodiment of this step (a), the method comprises the following sub-steps:

(a1) providing a suspension of red blood cells in at least 60 or 65% hematocrit,

(a2) measuring the osmotic fragility of RBC in this suspension,

(a3) a flow in counter-flow from the dialysis apparatus, in particular the dialysis tube, to the dissolution solution of the suspension, to adjust the flow rate of the suspension of the RBC or to adjust the flow rate of the dissolution solution, or Process for dissolution and internalization of the active ingredient as a function of osmotic fragility measured in (a2), comprising adjusting the osmotic pressure of

(a4) Process for sewing RBC.

In this embodiment, step (a1) comprises washing / centrifugation of cell residues and suspending RBC washed in physiological buffer in at least 60 or 65% hematocrit.

Preferably, a temperature of 2 to 8 ° C. is maintained during steps (a1) and (a3) and preferably the temperature of the product used is 2 to 8 ° C.

Preferably, the sewing process of the RBC is carried out using a hypertonic solution and preferably at a temperature of 30 to 40 ° C., in particular about 37 ° C.

After sewing, the RBCs are separated from the sewing medium using a separation process, preferably centrifugation. After centrifugation, the residue of RBC is collected in centrifuge tubes or containers. According to an advantageous feature of the invention, the collection consists of all or substantially all fractions that are likely to contain RBCs, thereby increasing the final cell yield after removal of subsequent fragile cells.

In one embodiment, the active ingredient is L-asparaginase. Other embodiments include the inclusion, preferably encapsulation, of the active ingredient selected from the following: IHP, ADI, Factor VIII, Factor IX, Alglucosidase, Beta-glucosidase, Bisphosphonates, especially second and third generations , Uricase, thymidine phosphorylase, adenosine deaminase and the like.

The RBC between steps (a) and (b) or after steps (c) or (d) further modifies or imparts functionality to the surface of the RBC by surface grafting or coupling that changes its properties. You can experience the treatment. In certain embodiments, this treatment is carried out after incubation, in particular after the incubation step or after the washing step following the incubation. This treatment may be one of the following:

Chemical treatment with agents that change the surface of the RBC, in particular bridging or crosslinking agents, for example bis (sulfosuccinimidyl) suverate (BS3), glutaraldehyde and neuroamidase (denaturant) ;

Heat treatment carried out, for example, under the following conditions: from about 42 to about 55 ° C., preferably from about de 47 to about 51 ° C. for about 15 to about 90 minutes, preferably about 25 to about 50 minutes. Heating the RBC at temperature;

Forming an immune complex, preferably comprising an antibody of the IgG sub-type; For example, anti-Rhesus antibodies, anti-glycoporin A antibodies and anti-CR1 antibodies (CR1 = type 1 complement receptor)

Another subject of the invention is an incubation step of a suspension of RBCs, particularly encapsulating the active ingredient, followed by removal of the incubation medium, preferably by washing to remove the incubation medium to stabilize the suspension of RBC or RBC. Is the use of. Preferably, this use further comprises the additional step of suspending the RBC in a preservation solution. Many of the more precise features described above apply to this subject of the invention.

In particular according to the properties, this incubation step is for at least 30 minutes, in particular for at least 1 h, at least 280 mOsmol / kg and especially from about 280 to about 380 mOsmol / kg, preferably about 290 Incubation of the suspension or residue containing a solution at an osmotic pressure of from about 330 mOsmol / kg.

In addition, in particular, according to the invention, this incubation may comprise one or more of the following properties:

The incubation step is carried out at a temperature of about 2 to about 39 ° C. for a sufficient time to ensure dissolution of the fragile RBCs;

The incubation step is carried out at low temperatures, in particular from about 2 to about 10 ° C., more specifically from about 2 to about 8 ° C., and from about 1 h to about 72 h, in particular from 6 h to about 48 h, preferably from about 19 h to about Lasts for 30 h of time;

The incubation step is carried out at a higher temperature of about 20 to about 39 ° C., in particular at ambient temperature (25 ° C. ± 5 ° C.) and from about 30 minutes to about 10 h, in particular from about 1 h to about 6 h, preferably about Lasts from 2 h to about 4 h; It is possible to work at temperatures much higher than the ambient temperature, but this can have a negative effect on the yield, P50 and / or 2,3-DPG content of the cells;

In the incubation step, the suspension has an initial hematocrit of 10 to 85%, in particular 40 to 80%; For example, it is possible to incubate the resulting residue from the isolate with 70 to about 85% hematocrit, or the diluted residue with about 40 to 70% hematocrit;

The incubation step comprises stirring the suspension;

The incubation step does not include stirring.

A further subject of the present invention is a stabilized suspension of RBCs encapsulating the active ingredient, which can be obtained by performing the method of the present invention.

In particular, the suspension in the preservation solution is 0.5 g / dl or less, in particular 0.3 g / dl or less, more specifically 0.2 g / dl or less, preferably 0.15 g / dl, at storage at a temperature of 2 to 8 ° C. in the 72 hour difference. characterized by extracellular hemoglobin levels maintained below dl, more preferably below 0.1 g / dl.

In particular, the suspension in the preservation solution is at most 0.5 g / dl, in particular at most 0.3 g / dl, more particularly at 0.2 g / d for storage for 24 to 20 days, especially for 24 to 72 hours and at temperatures of 2 to 8 ° C. characterized by extracellular hemoglobin levels maintained at or below dl, preferably at most 0.15 g / dl, more preferably at most 0.1 g / dl.

Extracellular hemoglobin levels are advantageously Clin. Biochem. 8, 96-102, G.B. Blakney and A.J. Measurements are made using the reference manual method described by Dinwoodie. In addition, automated equipment exists to enable this measurement at each of its own specific sensitivity. Nevertheless, it has been shown in the examples using three different methods that it is possible to obtain conforming levels by the method of the invention or that three methods can be used for this control.

In particular, it is characterized by a rate of hemolysis in which the suspension in the preservation solution is maintained at 2 or less, in particular 1.5 or less, preferably 1% or less at storage at a temperature of 2 to 8 ° C. in the 72 hour time difference.

In particular, it is characterized by a rate of hemolysis in which the suspension in the preservation solution is maintained at no more than 2%, in particular no more than 1.5%, preferably no more than 1% at temperatures of 24 to 20 days, especially 24 to 72 hours and at temperatures of 2 to 8 ° C. To have.

In particular, the hematocrit of the suspension is not more than 40%.

In one embodiment, the active ingredient is L-asparaginase. Other embodiments include the inclusion, preferably encapsulation, of the active ingredient selected from the following: IHP, ADI, Factor VIII, Factor IX, Alglucosidase, Beta-glucosidase, Bisphosphonates, especially second and third generations , Uricase, thymidine phosphorylase, adenosine deaminase and the like.

Advantageously, suspensions in preservation solutions are readily available, with low extracellular hemoglobin levels, especially in compliance with FDA recommendations.

A further subject of the invention is a method of treatment by injection of a suspension of RBCs encapsulating the active ingredient.

In a first embodiment of this method, the patient is given an injection of a suspension of RBCs encapsulating the active ingredient prepared 1-72 h, in particular 10-72 h, prior to injection. The suspension is at least 40% hematocrit. It is contained in a preservation solution. Extracellular hemoglobin levels are maintained at 0.5 g / dl or less, in particular at most 0.3 g / dl, more specifically at most 0.2 g / dl, preferably at most 0.15 g / dl, more preferably at most 0.1 g / dl, and And / or the rate of hemolysis is 2% or less, in particular 1.5% or less, preferably 1% or less. Suspensions are not subject to washing or similar prior to injection.

In another embodiment, the method comprises the steps of providing a cell residue, suspending in physiological buffer in at least 60 or 65% hematocrit, encapsulating the active ingredient in these RBCs using a lysis and sewing process, and obtaining Incubating the prepared RBC, washing the latter and collecting the final suspension of the RBC. The hematocrit of the suspension is at least 40%. It is contained in a preservation solution. This suspension is stored at a temperature of 2-8 ° C. This final suspension is injected in the patient 1 h to 72 h, preferably 24 to 72 h after preparation of the suspension. The extracellular hemoglobin level of this suspension is at most 0.5 g / dl, in particular at most 0.3 g / dl, more specifically at most 0.2 g / dl, preferably at most 0.15 g / dl, more preferably at most 0.1 g / dl, and And / or its hemolysis rate is 2% or less, in particular 1.5% or less, preferably 1% or less. Suspensions are not subject to pre-injection washes or the like.

This method particularly includes the following steps:

(a1) providing a suspension of red blood cells in at least 60 or 65% hematocrit,

(a2) measuring the osmotic fragility of RBC in this suspension,

(a3) a countercurrent flow of the suspension of RBC into the dissolution solution in the dialysis tube, adjusting the flow rate of the suspension of the RBC or adjusting the flow rate of the dissolution solution, or adjusting the osmotic pressure of the dissolution solution, ( dissolution and internalization of the active ingredient as a function of osmotic fragility measured in a2),

(a4) sewing process of RBC,

(b) optionally, one or more wash cycles by dilution of the suspension or residue obtained in solution (a4), a collection of residues or suspensions of RBC in the wash solution,

(c) incubating the residue or suspension of step (a4) or (b) as it is or after addition of the incubation solution,

(d) removing the liquid medium from the suspension incubated in step (c),

(e) suspending the RBC obtained in (d) in a solution which enables injection in a patient, in particular a preservation solution.

In one embodiment of this method, the RBCs contain L-asparaginase. In addition, the active ingredient may be one of the other active ingredients described above, but is not limited thereto.

The present invention will now be described in more detail with the aid of embodiments in which a single figure is given as a method described in EP 1 773 452 and a non-limiting example relating to the figure that illustrates the method of the present invention, for 72 hours extracellular. The results obtained for each of them in terms of hemoglobin are provided.

Example  1: NaCl + at ambient temperature for varying period Glucose  Incubation of Red Blood Cells

The residue of human RBC is treated in the presence or absence of the active ingredient (L-asparaginase) until the sewing is completed according to the method described in patent EP 1 773 452 and the suspension of RBC collected in pouche). Pouches of RBCs were transferred to a washer (Cobe 2991). The suspension was pre-dilute with 0.9% NaCl and 0.2% glucose and then transferred to the centrifugation pouche of the centrifuge. The suspension was centrifuged for 2 minutes at 3000 rpm. The supernatant was then directed to the waste pouch at a supernatant outflow rate set at 350 ml / min. The dilution / centrifugation operation was repeated two more times to terminate the wash cycle. Pouches of centrifuges containing 80% hematocrit RBCs were placed in the washer for 30 minutes, 1 hour or 3 hours at ambient temperature (24 ± 5 ° C). After incubation, another wash cycle was performed. The RBCs were then resuspended with 100 ml AS-3 (Caridian BCT) stock solution and stored at 5 ± 3 ° C. The stability of the product was determined by measuring extracellular hemoglobin on the day of preparation (DO), then 24 hours (D1), after 48 hours (D2) and the like. This measurement was performed using two methods: an automated analyzer (Cell Dyn Ruby: measurement at 555 nm, hemoglobin linearity 0.0-25.0 g / dl ± 0.3, coefficient of variation <2.0% Use of background noise to measure trace amounts of hemoglobin, improved sensitivity up to ± 0.2 g / dl; or Excell 2280 automated analyzer: measurement at 540 nm, hemoglobin linearity 1.5-30.0 g / dl ± 0.1 , Coefficient of variation (1%), or Clin. Biochem. 8, 96-102, G.B. Blakney and A.J. By visible spectrophotometry at 577 nm following the reference manual method described by Dinwoodie. In the examples the automated analyzer was used according to the manufacturer's recommendations. The results are provided below.

1.1 Determination of extracellular hemoglobin (en g / dl) performed using a Ruby analyzer on four suspensions of RBC encapsulating L-asparaginase:

Batch N ° DO D1 D2 D3 3 hours at ambient temperature Incubation PH- PF001 -130115-01 0.000 0.000 0.007 0.007 PH- PF001 -130115-02 0.015 0.022 0.022 0.007 PH- PF001 -130116-01 0.030 0.030 0.040 0.030 PH- PF001 -130116-02 0.000 0.007 0.007 0.013 PH- PF001 -130117-01 0.007 0.020 0.010 0.030

1.2 Determination of extracellular hemoglobin by spectrophotometry on three RBC suspensions that do not encapsulate L-asparaginase (ERY-ASP-121217-CG) or (ERY-121210-MA and ERY-121217-QB) (g / dl):

Batch N ° D0 D1 D2 D3 D8 3 hours at ambient temperature Incubation ERY -121210-MA 0.120 0.120 0.127 0.141 0.188 ERY -121217- QB 0.123 0.146 0.139 0.152 - ERY -ASP-121217-CG 0.084 0.077 0.081 0.098 -

1.3 Determination of Extracellular Hemoglobin Using Excel 2280 Analyzer on Two RBC Suspensions (g / dl): First suspension encapsulating L-asparaginase, second suspension without active ingredient:

Batch N ° D0 D1 D2 D3 D4 1 hour at ambient temperature ERY -ASP-130228-EC 0.11 - - - 0.18 30 minutes at ambient temperature GR - LR -130325- QB 0.12 0.11 0.12 0.12 0.12

This method comprising the step of incubating RBC in NaCl + glucose (of varying from 30 minutes to 3 hours) at 0.2 g / dl at D3 (72h) and longer term D8, i.e. up to 8 days after preparation of the product. A stable product with lower extracellular hemoglobin levels was obtained. This result was confirmed by the measurement of extracellular hemoglobin performed using three different methods.

Example  2: at ambient temperature Incubation  Changes in extracellular hemoglobin levels during

The residue of human RBC was treated as in Example 1 but without the active ingredient. Aliquots were taken during incubation at ambient temperature and centrifuged at 1000 ° C. for 10 minutes. Supernatants were collected and the level of hemoglobin at 577 nm was determined by visible spectrophotometry. The results are provided below:

At ambient temperature Incubation  Hours (h) 0 0.5 One 2 3 Hemoglobin in supernatant (g / dl) 1.418 1.848 2.063 2.793 3.089

The results indicate that the hemolysis of RBCs was constant for 3 hours of incubation time in NaCl + glucose at ambient temperature. Therefore, this incubation step provides a significant contribution to removing the most fragile RBCs and thus it is essential for the method to obtain a stable final product. Nevertheless, the incubation time can be reduced to 30 minutes as shown in Example 1. Even so, non-haemolysed RBCs, which are fragile during the incubation step, are weakened during this step and burst during the final wash.

Example  3: stock solution for 24 hours at 5 ± 3 ° C (AS-3 or SAG- Mannitol ) Of RBC of Incubation

The residue of the RBC was treated and included L-asparaginase until the sewing was completed according to the method described in patent EP 1 773 452. Pouches of RBCs were transferred to a washer (Cobe 2991). The suspension was pre-diluted with 0.9% NaCl and 0.2% glucose and then transferred to the pouch of the centrifuge. The suspension was centrifuged for 2 minutes at 3000 rpm. The supernatant was then directed to the waste pouch at a supernatant outflow rate of 350 ml / min. The dilution / centrifugation operation was repeated two more times to terminate the wash cycle. The suspension of RBC of 80% hematocrit was then resuspended with 100 ml AS-3 or 80 ml SAG-mannitol preservation solution. Pouches of ˜50% hematocrit of RBCs were then stored at 5 ± 3 ° C. for 24 hours and then washed before re-suspension in 100 ml AS-3 stock solution (Caridian BCT). The final product was stored at 2-8 ° C. The stability of the product was determined by measuring extracellular hemoglobin on the day of preparation (DO), after 24 hours (D1), after 48 hours (D2) and the like. This measurement was performed at 577 nm using visible spectrophotometry. The results are provided below:

Incubation  Time and temperature Incubation
solution Batch N ° D0 D1 D2 D3 D7 24h / 5 ± 3 ℃ SAG- Mannitol ERY -ASP-121211-CG-01 0.107 0.118 0.187 - 0.186 SAG- Mannitol ERY -ASP-121218-CG 0.06 0.073 0.088 - - AS-3 ERY -ASP-121218-MA 0.104 0.128 0.140 - -

Incubation of RBC in AS-3 or SAG-mannitol gave similar results. The product has extracellular hemoglobin levels of less than 0.2 g / dl for more than 48 hours and shows very good stability and similar results can be expected at D7 as shown for batch ERY-ASP-121211-CG-01.

Example  4: improving cell yield

The claimed method as described in Example 1 provides for improved stability of the product. However, this has a negative effect on the cell yield of this method (˜55% vs. ˜65% for the method described in EP 1 773 452). One of the wash parameters was modified during two cycles of the claimed method in order to maintain good stability of the product while obtaining better cell yield. In summary, under the conditions of Example 1 with the exception of the effluent rate of supernatant set to 100 ml / min instead of 350 ml / min, a batch was created during centrifugation of the cell suspension. This allowed the RBC detector to perform faster detection of the boundary between supernatant and RBC. The amount of RBC sent to the waste pouch was reduced and the cell yield of this method increased. The average yield for the method described in Example 4 is 73% vs 54% for the method of Example 1. The stability of the product is substantially the same.

Determination of extracellular hemoglobin (g / dl) and cell yield in% by Excell 2280 spectrophotometry:

D0 D1 D2 D3 Cell yield Method of EP1773452 0.3 ± 0.08 0.83 ± 0.22 1 ± 0.22 1.08 ± 0.22 ~ 65% (average of 189 batches) Method of Example 1 0.11 ± 0.02 0.11 ± 0.03 0.12 ± 0.03 0.13 ± 0.03 ~ 54 ± 3% (average of 5 batches) Method of Example 4 0.08 ± 0.02 0.13 ± 0.04 0.13 ± 0.06 0.15 ± 0.06 ~ 73 ± 5% (average of 9 batches)

Example  5: Reduction of extracellular hemoglobin levels after optimization of the preparation method

The extracellular hemoglobin levels found in the product prepared according to the method described in patent EP 1 773 452 are compared to that of the method of Example 1. Reduction of extracellular hemoglobin after 72 hours of storage time, since the method of EP 1 773 452 falls from 1.5 g / dl to less than 0.2 g / dl after optimization, ie due to a decrease in hemoglobin levels above 7 Is noted.

Claims (20)

A method of obtaining a stabilized suspension of RBC that encapsulates the active ingredient from a fully resealed red blood cell (RBC) suspension comprising the active ingredient, the method comprising an osmotic pressure of at least 280 mOsmol / kg For at least 30 minutes, or for 30 minutes to 3 hours, incubating the sewn RBCs in an incubation solution, wherein the incubation solution is a solution that does not contain an agent that denatures the RBC membrane, and then the liquid medium is Removing from the incubated suspension, and placing the obtained RBC in a solution that allows injection of the suspension in a patient,
Wherein said incubation solution does not contain bridging or cross-linking agents. The method of claim 1, comprising encapsulating the active ingredient by lysis-resealing prior to incubation and obtaining a stitched RBC comprising the active ingredient. The method of claim 1, wherein the incubation is performed at an osmotic pressure of 280 to 380 mOsmol / kg. The method of claim 1, wherein the incubation is performed at an osmotic pressure of 290 to 330 mOsmol / kg. The method of claim 1, wherein the incubation has a duration of at least 1 hour. The method of claim 1, further comprising washing the sewn RBC prior to the incubation. The method of claim 6, wherein the washing step comprises separating at least 280 mOsmol / kg of osmotic pressure after at least one wash cycle comprising diluting the sewn RBC in solution or suspending the sewn RBC. How. The method of claim 7, wherein the washing step comprises two or three wash cycles. The method of claim 1 comprising the following steps:
(a) encapsulating the active ingredient inside the RBC, including contacting with the storage medium, contacting the active ingredient, and sewing the RBC;
(b) obtaining a suspension or residue comprising RBC comprising said active ingredient and a solution of osmotic pressure of at least 280 mOsmol / kg;
(c) incubating the residue or suspension of step (b) as it is for at least 30 minutes, or for 30 minutes to 3 hours, or after addition of an osmotic incubation solution of at least 280 mOsmol / kg;
(d) removing the liquid medium from the suspension incubated in step (c);
(e) suspending the RBC obtained in (d) in a solution that allows injection of the suspension in a patient. 10. The residue or suspension of RBC according to claim 9, wherein step (b) is followed by one or more wash cycles by dilution of the suspension or residue obtained in (a) in an osmotic pressure of at least 280 mOsmol / kg. The method comprising the step of. The process according to claim 9 or 10, wherein in step (d) the separation, or dilution before separation, is carried out. The method of claim 1, wherein the incubation step is performed at a temperature of 2 to 39 ° C. 11. The method according to any one of claims 1 to 4, 9, and 10:
The incubation step is carried out at a low temperature of 2 to 10 ° C. and lasts from 1 hour to 72 hours; or
The incubation step is carried out at a temperature of 20 to 39 ° C. and lasts from 30 minutes to 1 hour. The method according to any one of claims 1 to 4, 9, and 10, after suspension in a preservation solution to an extracellular hemoglobin level or 2% or less, which is maintained at 0.5 g / dl or less at a temperature of 2 to 8 ° C. at a 72 hour difference. Recovering the stabilized RBC suspension with a maintained hemolysis rate. The extracellular hemoglobin level according to any one of claims 1 to 4, 9, and 10, which is maintained at 0.5 g / dl or less for 24 hours to 20 days and at a temperature of 2 to 8 ° C. after suspension in a preservation solution. Recovering a stabilized RBC suspension having a hemolysis rate maintained at or below%. The method according to any one of claims 1 to 4, 9, and 10, wherein the active ingredient is L-asparaginase, IHP, ADI, factor VIII, factor IX, alglucosidase, beta-glucosidase, bisphosphonate, urine Case, thymidine phosphorylase, adenosine deaminase. Stabilized suspension of RBCs encapsulating the active ingredient, which can be obtained by carrying out the process according to any one of claims 1 to 4, 9, and 10. The suspension according to claim 17, characterized by an extracellular hemoglobin level maintained below 0.5 g / dl at a temperature of 2 to 8 ° C., or a hemolysis rate maintained below 2% at 72 hours after suspension in a preservation solution. . 18. The method according to claim 17 characterized by an extracellular hemoglobin level maintained at 0.5 g / dl or less or a hemolysis rate maintained at 2% or less for 24 hours to 20 days after suspension in a preservation solution and at a temperature of 2 to 8 ° C. Suspension. The method according to claim 17, wherein the active ingredient is L-asparaginase, IHP, ADI, factor VIII, factor IX, alglucosidase, beta-glucosidase, bisphosphonate, uricase, thymidine phosphorylase, adenosine des Suspension which is chosen from aminase.

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